WO2018051863A1

WO2018051863A1 - Measurement method

Info

Publication number: WO2018051863A1
Application number: PCT/JP2017/032132
Authority: WO
Inventors: 野田　哲也; 史生長井; 洋一青木; 真紀子大谷
Original assignee: コニカミノルタ株式会社
Priority date: 2016-09-14
Filing date: 2017-09-06
Publication date: 2018-03-22
Also published as: EP3514522A4; EP3514522A1; JPWO2018051863A1; US20210285874A1; JP6760384B2; US11169089B2

Abstract

In the present invention, a measurement chip having a prism, a metal film, and a capture body is prepared. In a state in which a sample is present on the metal film, when a first light is radiated to the metal film at a first incidence angle less than a critical angle from the prism side, the first light transmitted through the metal film and the sample is scattered in the sample, and the scattered light thereby obtained is detected. In a state in which a substance to be measured is captured by the capture body and a sample is not present on the metal film, a signal indicating the amount of the substance to be measured is detected, the signal being generated by a measurement chip when a second light is radiated to the metal film at a second incidence angle equal to or greater than the critical angle from the prism side. A measurement value indicating the amount of the substance to be measured, the measurement value being determined from the signal, is corrected on the basis of the hematocrit value of the sample determined from the light quantity of the scattered light.

Description

Measuring method

The present invention relates to a measurement method for measuring the amount of a substance to be measured in a specimen containing whole blood using surface plasmon resonance.

In clinical examinations, if a very small amount of a substance to be measured such as protein or DNA in blood can be measured with high sensitivity and quantitativeness, it becomes possible to quickly grasp the patient's condition and perform treatment. Therefore, there is a need for a method that can measure a substance to be measured in blood with high sensitivity and quantitative.

Surface plasmon resonance (SPR) method and surface plasmon excitation enhanced fluorescence spectroscopy (Surface Plasmon-field enhanced Fluorescence Spectroscopy: below) (Abbreviated as “SPFS”). These methods use the fact that surface plasmon resonance (SPR) occurs when a metal film is irradiated with light under predetermined conditions (see, for example, Patent Documents 1 and 2). When SPR occurs in the metal film, plasmon scattered light having the same wavelength as the light irradiated to the metal film is emitted from the vicinity of the metal film.

In SPFS, a capture body (for example, a primary antibody) that can specifically bind to a substance to be measured is immobilized on a metal film to form a reaction field for specifically capturing the substance to be measured. When a specimen (for example, blood) containing a substance to be measured is provided in the reaction field, the substance to be measured is bound to the reaction field. Next, when a capture body (for example, a secondary antibody) labeled with a fluorescent substance is provided to the reaction field, the substance to be measured bound to the reaction field is labeled with the fluorescent substance. When the metal film is irradiated with excitation light in this state, the fluorescent substance that labels the substance to be measured is excited by the electric field enhanced by SPR and emits fluorescence. Therefore, the presence or amount of the substance to be measured can be detected by detecting the fluorescence. At this time, the plasmon scattered light is cut by the optical filter. In SPFS, a fluorescent substance is excited by an electric field enhanced by SPR, so that a substance to be measured can be measured with high sensitivity.

On the other hand, when measuring a substance to be measured in a liquid, the measured value is usually indicated by the mass of the substance to be measured per unit volume of the liquid, the amount of signal corresponding thereto, and the like. Therefore, when blood is used as the specimen, the measured value is indicated by the mass of the substance to be measured per unit volume of the liquid component (plasma or serum) in the blood, the signal amount corresponding thereto, and the like. Since the ratio of the liquid component in the blood varies from person to person, the measured value of whole blood (blood) cannot be uniformly converted into the measured value of the liquid component. Therefore, when whole blood is used as a specimen, the hematocrit value (ratio of blood cell volume in the blood) of the whole blood is measured, and the measured value of whole blood is measured using the hematocrit value as a liquid component (plasma or serum). It is necessary to convert to the measured value.

Conventional methods for measuring a hematocrit value include a micro hematocrit method for centrifuging blood and an electric conduction method for obtaining a hematocrit value from the electrical conductivity of blood. However, in the conventional method for measuring hematocrit, other devices such as a centrifuge, a device for measuring electrical conductivity, and a device for measuring hematocrit need to be newly prepared. Will increase.

On the other hand, in the measurement methods described in Patent Documents 1 and 2, when the excitation light is irradiated onto the metal film at an incident angle greater than the critical angle, it is generated at the measurement chip and scattered by the sample when passing through the sample. The hematocrit value of whole blood can be determined based on the amount of plasmon scattered light. For this reason, the measuring methods described in Patent Documents 1 and 2 do not require a new apparatus for measuring hematocrit values.

International Publication No. 2015/129615 International Publication No. 2016/039149

However, since the intensity (light quantity) of the plasmon scattered light is weak and the correlation between the hematocrit value and the intensity of the plasmon scattered light is also weak, the hematocrit value measurement method described in Patent Documents 1 and 2 has a higher hematocrit value. There is room for improvement from the viewpoint of measuring with high accuracy.

An object of the present invention is a measurement method using surface plasmon resonance, which can measure a hematocrit value with high accuracy without newly preparing a device for measuring a hematocrit value, and is to be measured in a specimen containing whole blood. It is to provide a measurement method capable of measuring the amount of a substance with high accuracy.

In order to solve the above problems, a measurement method according to an embodiment of the present invention is a measurement method for measuring the amount of a substance to be measured in a specimen containing whole blood using surface plasmon resonance. Preparing a measuring chip having a prism having an incident surface and a film-forming surface, a metal film disposed on the film-forming surface, and a capturing body fixed on the metal film; In the state where the specimen exists, when the first light is irradiated from the prism side at the first incident angle less than the critical angle, the first light that has passed through the metal film and the specimen is transmitted. A step of detecting scattered light obtained by scattering light in the specimen; and on the metal film, the substance to be measured is captured by the capture body, and the specimen is not present, Second incident angle greater than critical angle from prism side A step of detecting a signal indicating the amount of the substance to be measured, which is generated in the measurement chip when the second light is irradiated on the metal film, and the amount of the scattered light detected is determined from the detected amount of the scattered light. Correcting a measured value indicating the amount of the substance to be measured, determined from the detected signal, based on a hematocrit value.

According to the present invention, in a measurement method using surface plasmon resonance, a hematocrit value can be measured with high accuracy without adding a new hematocrit value measurement device. Therefore, according to the present invention, the amount of a substance to be measured in a specimen containing whole blood can be measured with high accuracy without increasing the manufacturing cost and measurement cost of the measuring apparatus.

FIG. 1 is a flowchart showing an example of a measurement method according to an embodiment of the present invention. FIG. 2 is a diagram illustrating an example of the configuration of a measurement chip and a measurement device (SPFS device) that can be used to perform the measurement method according to an embodiment of the present invention. 3A and 3B are graphs showing the results of simulation. 4A and 4B are graphs showing the results of Reference Experiment 1. FIG. 5A and 5B are graphs showing the results of Reference Experiment 1. FIG. FIG. 6 is a graph showing the correlation between the amount of scattered light and the hematocrit value. FIG. 7 is a graph showing the results of Reference Experiment 2. FIG. 8 is a graph showing the results of Reference Experiment 3. FIG. 9 is a flowchart illustrating an example of a measurement method according to the modification.

Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. The measurement method according to the present invention is a measurement method for measuring the amount of a substance to be measured in a specimen containing whole blood using surface plasmon resonance. Here, as a representative example of the measurement method according to the present invention, a measurement method using surface plasmon excitation enhanced fluorescence spectroscopy (Surface Plasmon-field enhanced Fluorescence Spectroscopy: hereinafter abbreviated as “SPFS”) will be described. In the measurement method according to the present embodiment, fluorescence emitted from a fluorescent substance that labels the substance to be measured is detected as a signal indicating the amount of the substance to be measured.

FIG. 1 is a flowchart showing an example of a measurement method according to the first embodiment. FIG. 2 is a diagram illustrating an example of the configuration of the measurement chip 10 and the measurement apparatus (SPFS apparatus) 100 that can be used to implement the measurement method according to the first embodiment. The measurement chip 10 and the SPFS device 100 will be described in detail separately.

The measurement method according to the present embodiment includes a step of preparing for measurement (step S110), a step of determining an enhancement angle (step S111), a step of measuring a first optical blank value (step S112), A step of measuring the second optical blank value (step S113), a step of performing a primary reaction (step S114), a step of detecting scattered light (step S115), and a step of performing a secondary reaction (step S116). And a step of detecting a fluorescent signal (step S117) and a step of correcting the measurement value (step S118).

1) Preparation for measurement First, preparation for measurement is performed (step S110). Specifically, the measurement chip 10 is prepared, and the measurement chip 10 is installed in the chip holder 142 arranged at the installation position of the SPFS device 100. Here, the “installation position” is a position for installing the measurement chip 10 in the SPFS device 100.

(Measurement chip)
Here, the measurement chip 10 used in the SPFS apparatus 100 will be described. As shown in FIG. 2, the measurement chip 10 includes a prism 20, a metal film 30, and a channel lid 40. In the present embodiment, the flow path lid 40 of the measurement chip 10 is integrated with a liquid chip 50 for containing a liquid.

The prism 20 has an incident surface 21, a film forming surface 22, and an exit surface 23. The incident surface 21 causes an outgoing light α from a light emitting unit 110 described later to enter the prism 20. A metal film 30 is disposed on the film formation surface 22. The exit surface 23 enters the prism 20 at the entrance surface 21, and causes the reflected light reflected by the interface (deposition surface 22) between the prism 20 and the metal film 30 to exit to the outside of the prism 20.

The shape of the prism 20 is not particularly limited. In the present embodiment, the shape of the prism 20 is a column having a trapezoidal bottom surface. The surface corresponding to one base of the trapezoid is the film formation surface 22, the surface corresponding to one leg is the incident surface 21, and the surface corresponding to the other leg is the emission surface 23.

The incident surface 21 is formed so that the emitted light α from the light emitting unit 110 is reflected by the incident surface 21 and does not return to the light source of the SPFS device 100. When the light source of the emitted light α is a laser diode (hereinafter also referred to as “LD”), when the emitted light α returns to the LD, the excited state of the LD is disturbed, and the wavelength and output of the emitted light α change. . Therefore, the angle of the incident surface 21 is set so that the emitted light α does not enter the incident surface 21 perpendicularly in a scanning range centered on an ideal resonance angle or enhancement angle.

Here, the “resonance angle” refers to the incident angle when the amount of reflected light of the emitted light α emitted from the emission surface 23 is minimized when the incident angle of the emitted light α with respect to the metal film 30 is scanned. means. The “enhancement angle” refers to scattered light having the same wavelength as the emitted light α emitted above the measurement chip 10 when the incident angle of the emitted light α with respect to the metal film 30 is scanned (hereinafter referred to as “plasmon scattered light”). It means the incident angle when the amount of γ is maximized. In the present specification, light emitted onto the measurement chip 10 when the incident angle of the emitted light α with respect to the metal film 30 is equal to or larger than the critical angle is referred to as “plasmon scattered light γ”. The light emitted above the measurement chip 10 when the incident angle of the emitted light α with respect to the metal film 30 is less than the critical angle is referred to as “scattered light γ ′”. In the present embodiment, the angle between the incident surface 21 and the film formation surface 22 and the angle between the film formation surface 22 and the emission surface 23 are both about 80 degrees.

Note that the resonance angle (and the enhancement angle near the pole) is generally determined by the design of the measurement chip 10. The design factors are the refractive index of the prism 20, the refractive index of the metal film 30, the thickness of the metal film 30, the extinction coefficient (extinction coefficient) of the metal film 30, the wavelength of the outgoing light α, and the like. The resonance angle and the enhancement angle are shifted by the substance to be measured trapped on the metal film 30, but the amount is less than several degrees.

The prism 20 is made of a dielectric that is transparent to the outgoing light α. The prism 20 has a number of birefringence characteristics. Examples of the material of the prism 20 include resin and glass. Examples of the resin constituting the prism 20 include polymethyl methacrylate (PMMA), polycarbonate (PC), and cycloolefin-based polymer. The material of the prism 20 is preferably a resin having a refractive index of 1.4 to 1.6 and a small birefringence.

The metal film 30 is disposed on the film formation surface 22 of the prism 20. As a result, surface plasmon resonance (hereinafter abbreviated as “SPR”) occurs between the photon of the outgoing light α incident on the film formation surface 22 under the total reflection condition and the free electrons in the metal film 30. Localized field light (generally also called “evanescent light” or “near field light”) can be generated on the surface of the metal film 30. The localized field light extends from the surface of the metal film 30 to a distance that is about the wavelength of the outgoing light α. The metal film 30 may be formed on the entire surface of the film formation surface 22 or may be formed on a part of the film formation surface 22. In the present embodiment, the metal film 30 is formed on the entire film formation surface 22.

A capture body for capturing the substance to be measured is immobilized on the metal film 30. A region where the capturing body is immobilized on the metal film 30 is particularly referred to as a “reaction field”. The capturing body may be fixed to the entire surface of the metal film 30 or may be fixed to a part of the surface. The capturing body specifically binds to the substance to be measured. For this reason, the substance to be measured can be immobilized on the metal film 30 via the capturing body.

The type of the capturing body is not particularly limited as long as the substance to be measured can be captured. For example, the capturing body is an antibody (primary antibody) or a fragment thereof that can specifically bind to the substance to be measured, an enzyme that can specifically bind to the substance to be measured, or the like.

The material of the metal film 30 is not particularly limited as long as it causes surface plasmon resonance (SPR) and at least a part of the emitted light α can be transmitted. Examples of the material of the metal film 30 include gold, silver, copper, and alloys thereof. The thickness of the metal film 30 is preferably 30 to 60 nm from the viewpoint of efficiently generating SPR and obtaining a desired transmittance for the emitted light α. The transmittance of the metal film 30 with respect to the emitted light α is, for example, 3 to 30% when the P-polarized light is irradiated onto the metal film 30 at an incident angle of 50 degrees.

Further, the thickness of the metal film 30 can be appropriately set according to the material from the above viewpoint. For example, when the material of the metal film 30 is gold, the thickness of the metal film 30 is preferably 30 to 55 nm in order to set the transmittance to 5 to 30% (see Reference Experiment 3 described later). When the material of the metal film 30 is silver, the thickness of the metal film 30 is preferably 35 to 60 nm in order to make the transmittance 3 to 20%. Further, when the material of the metal film 30 is copper, the thickness of the metal film 30 is preferably 30 to 55 nm in order to make the transmittance 5 to 25%. Among the above materials, the material of the metal film 30 is preferably gold. This is because gold has higher transmittance than other metals, can generate SPR with high efficiency, and has high stability (for example, oxidation resistance) against the external environment. In the present embodiment, the metal film 30 is a gold thin film. The method for forming the metal film 30 is not particularly limited. Examples of the method for forming the metal film 30 include sputtering, vapor deposition, and plating.

The flow path lid 40 is disposed on the metal film 30. When the metal film 30 is formed only on a part of the film formation surface 22 of the prism 20, the flow path lid 40 may be disposed on the film formation surface 22. In the present embodiment, the flow path lid 40 is disposed on the metal film 30. By placing the flow path lid 40 on the metal film 30, an accommodating portion for accommodating the liquid is formed on the metal film 30. In the present embodiment, the storage portion is a flow path 41 through which a liquid flows. The channel 41 has a bottom surface, a top surface, and a pair of side surfaces that connect the bottom surface and the top surface. In this specification, the surface of the channel 41 on the prism 20 side is referred to as the bottom surface of the channel, and the surface of the channel 41 that faces the bottom surface of the channel 41 is referred to as the top surface of the channel. The distance between the bottom surface of the flow channel 41 and the top surface of the flow channel 41 is referred to as the height of the flow channel 41.

A recess (flow channel groove) is formed on the back surface of the flow channel lid 40. The flow path lid 40 is disposed on the metal film 30 (and the prism 20), and the opening of the concave portion is closed by the metal film 30, whereby the flow path 41 is formed. From the viewpoint of sufficiently securing a region covered with the localized field light, it is preferable that the height of the flow channel 41 (depth of the flow channel groove) is large to some extent. From the viewpoint of reducing the amount of impurities mixed into the flow channel 41, the height of the flow channel 41 (the depth of the flow channel groove) is preferably small. From such a viewpoint, the height of the flow path 41 is preferably in the range of 0.05 to 0.15 mm. Both ends of the channel 41 are connected to an inlet and an exhaust port (not shown) formed in the channel lid 40 so that the inside of the channel 41 communicates with the outside.

The channel lid 40 is preferably made of a material that is transparent to light (fluorescence β, scattered light γ ′, and plasmon scattered light γ) emitted from the metal film 30. Examples of the material of the flow path lid 40 include glass and resin. Examples of the resin include polymethyl methacrylate resin (PMMA). Moreover, as long as it is transparent with respect to said light, the other part of the flow-path cover 40 may be formed with the opaque material. The flow path lid 40 is bonded to the metal film 30 or the prism 20 by, for example, adhesion using a double-sided tape or an adhesive, laser welding, ultrasonic welding, or pressure bonding using a clamp member.

In addition, you may use the flow path cover in which the recessed part (flow path groove) is not formed in the back surface instead of said flow path cover 40. FIG. In this case, the flow path lid having no recess and the metal film 30 or the prism 20 are formed by using a double-sided tape having a thickness of 0.05 to 0.15 mm in which a through hole serving as a flow path is formed in the central portion. Be joined. In this way, the flow path 41 may be formed.

The measurement chip 10 is usually replaced for each measurement. The measuring chip 10 is preferably a structure having a length of several mm to several cm for each piece, but is a smaller structure or a larger structure that is not included in the category of “chip”. May be.

In the step of preparing for measurement (step S110), when a storage reagent is present on the metal film 30 of the measurement chip 10, the capture body can appropriately capture the substance to be measured on the metal film 30. To remove the storage reagent.

2) Determination of enhancement angle Next, the enhancement angle is determined (step S111). Specifically, first, the measurement liquid is injected into the flow path 41. For example, the measurement liquid is provided in the flow path 41 using a pipette 131 described later. The measurement solution only needs to be transparent to the emitted light α. For example, phosphate buffered saline (PBS), Tween20-containing Tris buffered saline (TBS-T), HEPES buffered saline (HBS), etc. Buffer solution.

Next, the incident angle of the emitted light α with respect to the metal film 30 on the back surface of the metal film 30 corresponding to the region where the capturing body is fixed via the prism 20 in a state where the measurement liquid is present in the flow path 41. Irradiates the emitted light α while scanning, and detects the plasmon scattered light γ generated in the measurement chip 10. In the present embodiment, the angle adjustment mechanism 112 scans the incident angle of the emitted light α from the light source unit 111 to the metal film 30 and detects the plasmon scattered light γ by the light receiving sensor 126. Thereby, data including the relationship between the incident angle of the outgoing light α with respect to the metal film 30 and the light amount of the plasmon scattered light γ is obtained. The obtained data is analyzed to determine the enhancement angle, which is the incident angle when the amount of plasmon scattered light γ is maximized. In this step, the optical filter 124 (described later) that transmits only the fluorescent β component and removes the emitted light α component (scattered light γ ′ and plasmon scattered light γ) is partially or entirely plasmon scattered light γ. It is arranged to be located outside the optical path. Thereby, the emitted light α component enters the light receiving sensor 126.

The enhancement angle is determined by the material and shape of the prism 20, the thickness of the metal film 30, the refractive index of the liquid in the flow channel 41, etc., but the type and amount of the trapped body in the flow channel 41, the shape error of the prism 20 The measurement chip 10 varies slightly due to various factors such as an installation error in the SPFS device 100. For this reason, it is preferable to determine the enhancement angle every time measurement is performed. The enhancement angle is determined on the order of about 0.1 degree.

3) Measurement of first optical blank value Next, the first optical blank value is measured (step S112). Here, the first optical blank value is an optical blank value used when determining the hematocrit value, and is a predetermined first value less than the critical angle in a state where the measurement liquid is present in the flow path 41. It means the amount of light having the same wavelength as the emitted light α emitted above the measuring chip 10 when the emitted light α is irradiated onto the metal film 30 at an incident angle. Hereinafter, the emitted light α irradiated to the metal film 30 at the first incident angle is also referred to as “first emitted light α ₁ (referred to as“ first light ”in the claims”). .

In this step, first, the metal film 30 is irradiated with the _first outgoing light α1 at a _first incident angle less than the critical angle, and light emitted above the measurement chip 10 is detected. Although the details will be described later, the first incident angle only needs to be less than the critical angle. From the viewpoint of measuring the hematocrit value with high accuracy and suppressing the SPFS device 100 from becoming large, the first incident angle. Is preferably smaller than the critical angle (for example, about 5 to 10 degrees). Further, in the present embodiment, while the light source unit 111 is irradiated with the first outgoing light alpha ₁ to the metal film 30 in the first incidence angle, detecting the light emitted above the measuring chip 10 by the light receiving sensor 126 To do. Thereby, the first optical blank value, which is the amount of light that becomes noise in the detection of the scattered light γ ′ (step S115), is obtained. Even in this step, the optical filter 124 is disposed so that a part or all of the optical filter 124 is located outside the optical path.

4) Measurement of second optical blank value Next, a second optical blank value is measured (step S113). Here, the second optical blank value is an optical blank value used when determining the amount of the substance to be measured, and in a state in which the measurement liquid is present in the flow path 41, the second optical blank value is a predetermined value greater than the critical angle. It means the amount of light having the same wavelength as the emitted light α emitted above the measuring chip 10 when the emitted light α is irradiated onto the metal film 30 at the second incident angle. Hereinafter, the emitted light α irradiated to the metal film 30 at the second incident angle is also referred to as “second emitted light α ₂ (referred to as“ second light ”in the claims)”. .

In this step, first, the incident angle of the emitted light α with respect to the metal film 30 (deposition surface 22) is switched to the second incident angle. The second incident angle only needs to be equal to or greater than the critical angle, and is an angle for generating SPR in the metal film 30 irradiated with the emitted light α. In the present embodiment, the light source in the light source unit 111 is rotated to switch from the first incident angle to the enhancement angle (second incident angle) determined in step S111. Next, the metal film 30 is irradiated with the _second outgoing light α2 at an enhancement angle, and the light emitted above the measurement chip 10 is detected. In this embodiment, while irradiating the metal film 30 from the light source unit 111 and the second outgoing light alpha ₂ in enhancing angle, it detects the light emitted above the measuring chip 10 by the light receiving sensor 126. Thereby, the 2nd optical blank value which is the light quantity of the light used as noise in detection of a fluorescence signal (process S117) is obtained. In this step, the optical filter 124 is disposed on the optical path.

5) Primary reaction Next, the substance to be measured in the specimen is reacted with the capturing body on the metal film 30 (primary reaction; step S114). Specifically, first, the measurement liquid is removed from the flow path 41 and the specimen is injected into the flow path 41. For example, the sample is provided in the flow channel 41 after the measurement liquid in the flow channel 41 is aspirated using the pipette 131. Thereby, when the substance to be measured exists in the specimen, at least a part of the substance to be measured can be captured by the capturing body on the metal film 30.

The specimen contains whole blood and may be diluted as necessary. From the viewpoint of measuring scattered light γ 'with high intensity in step S115 described later, the concentration of the specimen is preferably high. This is because the amount of light scattered in the specimen increases as the concentration of the specimen increases, and the quantity of scattered light γ 'detected increases. On the other hand, the concentration of the sample is preferably low to some extent from the viewpoint of measuring the fluorescence β with high accuracy in step S117 described later. This is because when the concentration of the specimen is low to some extent, the amount of impurities in the specimen that are adsorbed (non-specific adsorption) on the capturing body can be reduced, and noise can be reduced. Moreover, it can suppress that the quantity of the to-be-measured substance which can be capture | acquired with a capture body will be saturated by adjusting the quantity of the to-be-measured substance with respect to the quantity of a capture body in the suitable range. As the diluent, for example, physiological saline can be used. Examples of substances to be measured in whole blood include troponin, myoglobin, and creatine kinase-MB (CK-MB).

6) Detection of scattered light Next, scattered light γ ′ indicating the hematocrit value of the specimen is detected (step S115). More specifically, when irradiated with the first outgoing light alpha ₁ to the metal film 30 in the first incidence angle from the prism 20 side, the first outgoing light alpha ₁ is transmitted through the metal film 30 and the specimen, the specimen Scattered light γ ′ obtained by scattering in is detected. More specifically, the scattered light gamma 'is obtained by first outgoing light alpha ₁ is scattered by the blood cells in the specimen. In the present embodiment, the light receiving sensor 126 detects the scattered light γ ′ while irradiating the metal film 30 with the emitted light α from the light source unit 111 so as to have the first incident angle. Thereby, the light quantity of the scattered light γ ′ indicating the hematocrit value of the specimen can be measured. In this step, the optical filter 124 is disposed such that a part or all of the optical filter 124 is located outside the optical path of the scattered light γ ′.

In this step, the first outgoing light alpha ₁ is the light of the same wavelength and the light quantity and the outgoing light alpha irradiated on the metal film 30 in step S112. The first outgoing light α ₁ irradiated to the metal film 30 is preferably P-polarized light having a wavelength of 600 to 700 nm. When the wavelength of the first outgoing light α ₁ is 600 to 700 nm, the transmittance of the first outgoing light α ₁ with respect to the gold film (metal film 30) is increased, and the scattered light γ ′ is hemoglobin in the specimen. It is possible to suppress the absorption by. As a result, the amount of scattered light γ ′ detected by the light receiving sensor 126 can be increased. Further, since the first outgoing light α ₁ is P-polarized light, the transmittance of the first outgoing light α ₁ with respect to the metal film 30 is further increased, so that the amount of the scattered light γ ′ to be detected is increased. Can do. As a result of these, the hematocrit value can be determined with high accuracy.

In this step, the first outgoing light alpha ₁ increases the amount of the resulting scattered light gamma 'by being scattered in the specimen, from the viewpoint of determining the hematocrit value with high precision, the concentration of the analyte is high that Is preferred. For example, the specimen is preferably whole blood having a dilution ratio of 1 to 10 times, and more preferably whole blood having a dilution ratio of 1 to 3 times (see Reference Experiment 2 described later). In the present embodiment, the specimen is whole blood having a dilution ratio of 1 to 10 times.

Also, as described above, from the viewpoint of determining the hematocrit value with high accuracy, it is preferable that the first incident angle is somewhat smaller than the critical angle. For example, it is preferable that the first incident angle is equal to or smaller than an angle that is 5 degrees smaller than the critical angle (see simulation described later). Furthermore, from the viewpoint of shortening the switching time of the first incident angle and the second incident angle and reducing the size of the SPFS device 100 by reducing the difference between the first incident angle and the second incident angle, The difference between the first incident angle and the second incident angle is preferably small. For example, the first angle of incidence may be equal to or greater than an angle that is 5 degrees less than the critical angle and 10 degrees less than the critical angle. More preferred.

Heretofore, an example in which the detection of scattered light γ ′ (step S115) is performed after the completion of the primary reaction (step S114) has been described, but the scattered light γ is started after the start of the primary reaction and before the end. 'May be detected. For example, the scattered light γ ′ is detected immediately after the sample is injected into the flow channel 41 after the start of the primary reaction step, and then the remaining reaction time until the reaction time of the primary reaction reaches a predetermined time. A primary reaction may be performed. In the primary reaction, the red blood cells may settle while the sample is being reciprocated in the flow path 41, and the scattered light γ ′ may not be detected accurately. This is preferable because the scattered light γ ′ can be detected before settling. This method is particularly effective when the red blood cells are likely to settle, such as when the reaction time of the primary reaction is long, when the dilution rate of the specimen is high, or when the hematocrit value is low. Of course, even when the scattered light γ ′ is detected prior to the primary reaction, the specimen is injected into the flow path 41, and the measured substance in the specimen and the capture body on the metal film 30 come into contact with each other. From this, the primary reaction itself is started. However, in the case of detecting the scattered light γ ′ during the primary reaction, from the viewpoint of accurately measuring the intensity of the scattered light γ ′, when detecting the scattered light γ ′, It is preferable to stop the movement of the fluid, that is, stop the liquid feeding. In any case, since the primary reaction can be performed in parallel with the detection of the scattered light γ ′, the total measurement time can be shortened, and one type of specimen can be prepared. It becomes simple.

7) Secondary reaction Next, the substance to be measured captured by the capturing body on the metal film 30 is labeled with a fluorescent material (secondary reaction; step S116). Specifically, first, the sample is removed from the flow path 41 by the pipette 131, and then the flow path 41 is washed with a buffer solution or the like to remove substances not captured by the capturing body. Next, a fluorescent labeling solution is provided in the flow path 41 by the pipette 131. Thereby, the substance to be measured can be labeled with the fluorescent substance. The fluorescent labeling solution is, for example, a buffer solution containing an antibody (secondary antibody) labeled with a fluorescent substance. Thereafter, the inside of the flow path 41 is washed with a buffer solution or the like to remove free fluorescent substances.

8) Detection of fluorescent signal Next, fluorescent β (signal) indicating the amount of the substance to be measured is detected (step S117). Specifically, on the metal film 30, the to-be-measured substance is captured by the capturing body and the sample is not present (the state where the flow path 41 is filled with the measuring solution), the enhancement angle (the first angle) from the prism 20 side. 2 angle of incidence), when the captor through the prism 20 is irradiated with the second outgoing light alpha ₂ on the rear surface of the metal film 30 corresponding to the area which has been immobilized, resulting in the measurement chip 10, the Fluorescence β (signal) indicating the amount of the measurement substance is detected. At this time, the second outgoing light α ₂ is excitation light that can directly or indirectly excite the fluorescent substance. In the present embodiment, the light-receiving sensor 126 detects the fluorescence β while irradiating the metal film 30 with the emitted light α from the light source unit 111 such that the second incident angle becomes an enhancement angle. Thereby, the fluorescence value which is the light quantity of fluorescence (beta) which shows the quantity of to-be-measured substance in a test substance can be measured with high intensity | strength. In this step, the optical filter 124 is disposed on the optical path.

In this specification, “the state in which no sample exists” means a state in which an operation for removing the sample from the flow channel 41 is performed. In other words, it is sufficient that the sample does not substantially exist in the flow channel 41, and a slight amount of the sample that cannot be removed may remain in the flow channel 41.

In this step, the second outgoing light alpha ₂ is the light of the same wavelength as the emitted light alpha that in step S111 and step S113 is irradiated onto the metal film 30. The second outgoing light α ₂ is light having a wavelength that can excite a fluorescent substance that labels the substance to be measured. Second outgoing light alpha ₂ wavelength and the light quantity may be the same as the first outgoing light alpha ₁ wavelength and light intensity, may be different. From the viewpoint of miniaturization of the measuring apparatus by use of the same light source, the second outgoing light alpha ₂ wavelength and the light quantity is preferably the same as the first outgoing light alpha ₁ wavelength and light intensity. In the present embodiment, the wavelength and light amount of the _second outgoing light α ₂ are the same as the wavelength and light amount of the first outgoing light α ₁ .

9) Correction of measurement value Next, the measurement value is corrected (step S118). Specifically, it indicates the amount of the substance to be measured, which is determined from the fluorescence β detected in step S117, based on the hematocrit value of the specimen, which is determined from the amount of scattered light γ ′ detected in step S115. Correct the measured value.

First, the hematocrit value of the specimen is determined from the amount of scattered light γ ′. The scattered light γ ′ includes a scattering component (signal component) caused by scattering in the specimen and a noise component (for example, prism 20, metal film 30, and flow path lid 40) caused by scattering in a region other than the specimen. First optical blank value). Therefore, the scattering component (signal component) in the specimen can be calculated by subtracting the first optical blank value obtained in step S112 from the light amount of the scattered light γ ′ detected in step S115. Next, the hematocrit value of the specimen can be determined based on the signal component and a calibration curve indicating the relationship between the amount of scattered light γ ′ and the hematocrit value.

Next, a measurement value indicating the amount (concentration) of the substance to be measured in the specimen is determined from the fluorescence value, which is the amount of fluorescence β. The fluorescence value includes a fluorescence component (signal component) derived from a fluorescent material that labels the substance to be measured, and a noise component (second optical blank value) caused by factors other than the fluorescent material. Therefore, by subtracting the second optical blank value obtained in step S113 from the fluorescence value obtained in step S117, a measurement value (signal component) indicating the amount of the substance to be measured in the sample can be calculated. it can.

Finally, the measurement value indicating the amount of the substance to be measured in the specimen is corrected based on the hematocrit value. Specifically, the measurement value is converted to the amount of the substance to be measured in the plasma by multiplying by the conversion coefficient c represented by the following formula (1).

[In the above formula (1), Hct is the hematocrit value (0 to 100%), and df is the dilution ratio of the specimen. ]

The amount (concentration) of the substance to be measured in plasma can be determined by the above procedure.

(simulation)
A simulation was performed in order to investigate a preferable range of the incident angle of the outgoing light α with respect to the metal film 30 when the scattered light γ ′ was detected in order to determine the hematocrit value. Specifically, for the metal films 30 having different thicknesses, a simulation was performed on the relationship between the incident angle of the emitted light α with respect to the metal film 30 and the reflectance (transmittance) of the emitted light α. In this simulation, the wavelength of the outgoing light α is 660 nm, the refractive index of the prism 20 is 1.528, the refractive index of the metal film 30 is 0.2144, the extinction coefficient of the metal film 30 is 3.85, and the refractive index of the specimen is set. Set to 1.331.

3A and 3B are graphs showing the results of simulation. 3A is a graph showing the relationship between the incident angle of the outgoing light α and the reflectance of the outgoing light α with respect to the metal film 30, and FIG. 3B shows the incident angle of the outgoing light α and the metal film 30 of the outgoing light α. It is a graph which shows the relationship with the transmittance | permeability with respect to. 3A, the horizontal axis indicates the incident angle (°) of the outgoing light α with respect to the metal film 30, and the vertical axis indicates the reflectance of the outgoing light α with respect to the metal film 30. In FIG. 3B, the horizontal axis indicates the incident angle (°) of the outgoing light α with respect to the metal film 30, and the vertical axis indicates the transmittance of the outgoing light α with respect to the metal film 30. 3A and 3B, the simulation result when the thickness of the metal film 30 is 30 nm is shown by a solid line, the simulation result when the thickness of the metal film 30 is 40 nm is shown by a dotted line, and the thickness of the metal film 30 is 50 nm. The simulation result is shown by a one-dot chain line, and the simulation result when the thickness of the metal film 30 is 60 nm is shown by a two-dot chain line.

As shown in FIG. 3A, the reflectance of the emitted light α with respect to the metal film 30 is such that the incident angle of the emitted light α is small in the region where the incident angle is about 55 degrees to about 60.5 degrees (critical angle). As it gets smaller. At this time, it can be seen that the amount of change in reflectance is larger as the incident angle is closer to the critical angle and smaller as the incident angle is farther from the critical angle. It can be seen that the reflectance hardly changes in the region where the incident angle is about 55 degrees or less.

As shown in FIG. 3B, the transmittance of the outgoing light α increases as the incident angle of the outgoing light α decreases in the region where the incident angle is about 55 degrees to about 60.5 degrees (critical angle). It has become. At this time, it can be seen that the amount of change in transmittance is larger as the incident angle is closer to the critical angle and smaller as the incident angle is farther from the critical angle. It can be seen that the transmittance hardly changes in the region where the incident angle is about 55 degrees or less.

From the results of this simulation, in order to determine the hematocrit value with high accuracy, even if the incident angle of the outgoing light α changes, the scattered light γ ′ is changed in a region where the change in reflectance (transmittance) can be suppressed. It can be seen that detection is preferable. From such a point of view, the first incident angle is preferably equal to or smaller than an angle smaller than the critical angle by 5 degrees. As a result, the critical angle changes due to a slight shift in the refractive index of the liquid in the flow channel 41 (for example, Δn = 0.001), or a slight shift in the incident angle of the outgoing light α (for example, 0.1 degree). It is possible to suppress a change in the transmitted light amount of the emitted light α through the metal film 30 due to the above. As a result, a change in the amount of the scattered light γ ′ to be detected can be suppressed, and the hematocrit value can be determined with high accuracy.

(Reference Experiment 1)
To compare the amount of scattered light γ 'with the amount of plasmon scattered light γ, and to compare the correlation between the amount of scattered light γ' and the hematocrit value, and the correlation between the amount of plasmon scattered light γ and the hematocrit value The experiment was conducted.

In Reference Experiment 1, the incident angle of the emitted light α with respect to the metal film 30 is scanned in a state where the measurement liquid, plasma, blood having a hematocrit value of 20%, or blood having a hematocrit value of 40% is present in the channel 41. While irradiating the metal film 30 with the emitted light α, the light emitted above the measuring chip 10 was detected by the light receiving sensor 126, and the amount of the light was measured. At the same time, the reflected light of the outgoing light α was detected by a light receiving sensor (not shown) to determine the reflectance of the outgoing light α.

4A and 4B and FIGS. 5A and 5B are graphs showing the results of Reference Experiment 1, in which the incident angle of the emitted light α with respect to the metal film 30, the amount of scattered light γ ′, the amount of plasmon scattered light γ, and the emitted light are shown. It is a graph which shows the relationship with the reflectance of (alpha). In FIGS. 4A and B and FIGS. 5A and B, the amount of light in the angle range where the incident angle is smaller than the critical angle (left side of the broken line in FIGS. 4A, B and 5A and B) means the amount of scattered light γ ′. The amount of light in the angle range (the right side of the broken line in FIGS. 4A, B and FIGS. 5A, B) where the angle is greater than or equal to the critical angle means the amount of plasmon scattered light γ. 4A is a measurement result in a state where a measurement liquid is present in the flow path 41, FIG. 4B is a measurement result in a state where plasma is present in the flow path 41, and FIG. FIG. 5B is a measurement result in a state where blood having a hematocrit value of 40% exists in the flow path 41. FIG. 4A and 4B and FIGS. 5A and 5B, the horizontal axis indicates the incident angle (°) of the outgoing light α with respect to the metal film 30, and the left vertical axis indicates the light quantity (count) of the scattered light γ ′ or the plasmon scattered light γ. The vertical axis on the right side shows the reflectance of the outgoing light α. Further, in FIGS. 4A and 4B and FIGS. 5A and 5B, the light amounts of the scattered light γ ′ and the plasmon scattered light γ are indicated by white symbols (□, ◇, Δ, and ○), and the reflectance of the emitted light α is black. The symbols (■, ◆, ▲, and ●) indicate.

In the graph showing the reflectance of the outgoing light α, a downwardly convex peak having a minimum value near 72 degrees is observed in a region where the incident angle is 61 degrees or more. This indicates that surface plasmon resonance occurs in the metal film 30 in a region where the incident angle is 61 degrees or more. Regardless of whether blood cells are present in the specimen, the amount of plasmon scattered light γ hardly changes in the region where the incident angle is 61 degrees or more (see FIGS. 4A, B and FIGS. 5A, B). .

On the other hand, as is apparent from the graph of the reflectance of the outgoing light α, the reflectance starts to decrease as the incident angle of the outgoing light α decreases with the vicinity of 61 degrees as a boundary. This indicates that the vicinity of 61 degrees is the critical angle. When the sample is a measurement solution or plasma and no blood cells are present in the sample, the amount of scattered light γ ′ hardly changes in a region where the incident angle is less than 61 degrees (see FIGS. 4A and B). On the other hand, when the specimen is blood and blood cells are present in the specimen, the amount of scattered light γ ′ increases in a region where the incident angle is less than 61 degrees (see FIGS. 5A and 5B). These results indicate that the outgoing light α is scattered by the blood cell component in the specimen when passing through the specimen. 5A and 5B, it can be seen that the intensity of the scattered light γ ′ caused by the blood cell component in the specimen is stronger than the intensity of the plasmon scattered light γ.

Next, the correlation between the light amount of the scattered light γ ′ and the hematocrit value is compared with the correlation between the light amount of the plasmon scattered light γ and the hematocrit value. FIG. 6 is a graph showing the correlation between the amount of scattered light γ ′ or plasmon scattered light γ and the hematocrit value. FIG. 6 summarizes the graph showing the relationship between the incident angle of the emitted light α with respect to the metal film 30 and the amount of scattered light γ ′ or plasmon scattered light γ in FIGS. 4A and 4B and FIGS. 5A and 5B into one graph. It is a graph. □ is the measurement result (FIG. 4A) in the state where the measurement liquid is present in the flow path 41, ◇ is the measurement result (FIG. 4B) in the state where the plasma is present in the flow path 41, and Δ is the flow path 41. FIG. 5A is a measurement result in a state where blood having a hematocrit value of 20% is present in the inside (FIG. 5A), and ○ is a measurement result in a state where blood having a hematocrit value is 40% is present in the flow path 41. . As shown in FIG. 6, the greater the hematocrit value in the specimen, the greater the amount of scattered light γ ′ in the region where the incident angle is less than 61 degrees. On the other hand, even if the hematocrit value in the specimen increases, the amount of plasmon scattered light γ in the region where the incident angle is 61 degrees or more hardly changes. That is, as is apparent from FIG. 6, it can be seen that the correlation between the amount of scattered light γ ′ and the hematocrit value is stronger than the correlation between the amount of plasmon scattered light γ and the hematocrit value.

From the results of Reference Experiment 1, the intensity of the scattered light γ ′ is higher than that of the plasmon scattered light γ, and the scattered light γ ′ is compared with the correlation between the amount of plasmon scattered light γ and the hematocrit value. It can be seen that the correlation between the amount of light and the hematocrit value is stronger. Therefore, it can be seen that the hematocrit value can be determined with higher accuracy by detecting the scattered light γ 'instead of the plasmon scattered light γ when determining the hematocrit value.

(Reference Experiment 2)
Experiments were conducted to determine the preferred range of specimen dilution ratios when determining hematocrit values. In Reference Experiment 2, all 5 types of hematocrit values (0%, 20%, 30%, 45% and 65%) diluted to 1-fold (no dilution), 3-fold or 15-fold were used. Blood was used as a specimen. When the measurement liquid is present in the flow path 41 and the metal film 30 is irradiated with the outgoing light α at an incident angle of 52 degrees, the scattered light γ ′ emitted above the measurement chip 10 is detected, An optical blank value of 1 was measured. Next, when the sample film is present in the channel 41 and the emitted light α is irradiated to the metal film 30 at an incident angle of 52 degrees, the scattered light γ ′ emitted above the measurement chip 10 is detected, The amount of scattered light γ ′ was measured. By subtracting the first optical blank value from the measured amount of scattered light γ ′, a scattering component (signal component) due to scattering in the specimen was calculated.

FIG. 7 is a graph showing the results of Reference Experiment 2, and is a graph showing the relationship between the specimen dilution rate and the correlation between the amount of scattered light γ ′ and the hematocrit value. In FIG. 7, the horizontal axis represents the hematocrit value Hct (%), and the vertical axis represents the signal component (count) of the scattered light γ ′. The results when the sample dilution rate is 1 (no dilution) are indicated by black circles (●), and when the sample dilution rate is 3 times, the results are indicated by black squares (■). The results when the dilution factor is 15 times are indicated by black triangles (▲).

As shown in FIG. 7, the slope of the graph increases as the dilution ratio of the specimen decreases (the specimen concentration increases). That is, it can be seen that the smaller the sample dilution ratio (the higher the sample concentration), the stronger the correlation between the amount of scattered light γ ′ and the hematocrit value. Therefore, when the scattered light γ ′ is detected to determine the hematocrit value, the concentration of the specimen is preferably high from the viewpoint of determining the hematocrit value with high accuracy. For example, the specimen is preferably whole blood having a dilution ratio of 1 to 10 times, and more preferably whole blood having a dilution ratio of 1 to 3 times.

(Reference Experiment 3)
An experiment was conducted to investigate a preferable range of the thickness of the metal film 30. In this experiment, for the metal film 30 having various thicknesses (10 to 80 nm), when the metal film 30 is irradiated with the emitted light α having a wavelength of 660 nm while changing the incident angle, the light is emitted from the emission surface 23 of the measurement chip 10. The reflected light of the emitted light α is detected by a light receiving sensor (not shown), and surface plasmon resonance is generated based on the amount of reflected light at a resonance angle that minimizes the amount of reflected light of the emitted light α. Efficiency was calculated. A gold film was used as the metal film 30.

FIG. 8 is a graph showing the results of Reference Experiment 3. In FIG. 8, the horizontal axis indicates the thickness (nm) of the metal film 30, and the vertical axis indicates the generation efficiency of surface plasmon resonance calculated based on the amount of reflected light at the resonance angle.

As shown in FIG. 8, the generation efficiency of the surface plasmon resonance is maximum when the thickness of the metal film 30 is around 40 nm. Therefore, when the metal film 30 is a gold film, when the thickness of the metal film 30 is, for example, 30 to 55 nm, a signal indicating the amount of the substance to be measured can be detected with high intensity, and the amount of the substance to be measured can be reduced. It can be seen that the measured values shown can be determined with high accuracy.

Further, when the thickness of the metal film 30 is 30 to 55 nm, the transmittance of the emitted light α to the metal film 30 is 5 to 30% (see FIG. 3B). That is, the scattered light γ ′ can be detected with high intensity by the high transmittance of the emitted light α with respect to the metal film 30. From this point of view, the hematocrit value can be determined with high accuracy, and the amount of the substance to be measured in the sample can be determined with high accuracy.

(SPFS device)
Next, an example of an SPFS apparatus for performing the measurement method according to this embodiment will be described. FIG. 2 is a configuration diagram illustrating an example of the configuration of the SPFS device 100. The SPFS device 100 includes a light emitting unit 110, a light detecting unit 120, a liquid feeding unit 130, a conveying unit 140, and a control processing unit (processing unit) 150. The SPFS apparatus 100 is used in a state where the above-described measurement chip 10 is mounted on a chip holder (holder) 142 of the transport unit 140.

The light emitting unit 110 emits outgoing light α (first outgoing light α ₁ and second outgoing light α ₂ ). In step S112 and step S115 described above, the light emitting unit 110 emits the emitted light α so as to enter the metal film 30 at the first incident angle. Moreover, in said process S113 and process S117, the light emission part 110 radiate | emits the emitted light (alpha) so that it may inject into the metal film 30 with a 2nd incident angle. That is, the first incidence angle, in order to determine the hematocrit value, which is the first incidence angle of the light emitted alpha ₁ of irradiating the metal film 30. On the other hand, the second angle of incidence, in order to determine a measurement value indicative of the amount of substance to be measured, a second incident angle of the light emitted alpha ₂ of irradiating the metal film 30. The first incident angle is less than the critical angle, and the second incident angle is greater than or equal to the critical angle.

When detecting the scattered light γ ′, the light emitting unit 110 emits the P wave for the metal film 30 toward the incident surface 21 at the first incident angle. The first outgoing light α _{1 at} this time passes through the film formation surface 22 and is emitted above the prism 20. Further, when detecting the fluorescence β or the plasmon scattered light γ, the light emitting unit 110 causes the P wave to the metal film 30 to be incident on the incident surface 21 at the second incident angle so that surface plasmon resonance occurs in the metal film 30. Exit toward. At this time, when the second outgoing light α ₂ is irradiated through the prism 20 at an angle at which surface plasmon resonance occurs on the metal film 30, localized field light that excites the fluorescent material is emitted on the surface of the metal film 30. To cause.

The light emitting unit 110 includes a light source unit 111, an angle adjustment mechanism 112, and a light source control unit 113.

The light source unit 111 emits collimated light having a constant wavelength and light amount so that the shape of the irradiation spot on the back surface of the metal film 30 is substantially circular. The light source unit 111 includes, for example, a light source, a beam shaping optical system, an APC mechanism, and a temperature adjustment mechanism (all not shown).

The type of the light source is not particularly limited, and is, for example, a laser diode (LD). Other examples of light sources include laser light sources such as light emitting diodes and mercury lamps. The wavelength of the outgoing light α emitted from the light source is, for example, in the range of 400 nm to 1000 nm. When the emitted light α emitted from the light source is not a beam, the emitted light α is converted into a beam by a lens, a mirror, a slit, or the like. When the emitted light α emitted from the light source is not monochromatic light, the emitted light α is converted into monochromatic light by a diffraction grating or the like. Furthermore, when the outgoing light α emitted from the light source is not linearly polarized light, the outgoing light α is converted into linearly polarized light by a polarizer or the like.

The beam shaping optical system includes, for example, a collimator, a band pass filter, a linear polarization filter, a half-wave plate, a slit, and a zoom means. The beam shaping optical system may include all of these or a part thereof.

The collimator collimates the outgoing light α emitted from the light source.

The band-pass filter turns the outgoing light α emitted from the light source into a narrow band light having only the center wavelength. This is because the outgoing light α emitted from the light source has a slight wavelength distribution width.

The linear polarization filter converts the outgoing light α emitted from the light source into linearly polarized light.

The half-wave plate adjusts the polarization direction of the light so that the P wave component is incident on the metal film 30.

The slit and zoom means adjust the beam diameter, contour shape, and the like of the emitted light α emitted from the light source so that the shape of the irradiation spot on the back surface of the metal film 30 is a circle of a predetermined size.

The APC mechanism controls the light source so that the output of the light source is constant. More specifically, the APC mechanism detects the amount of light branched from the emitted light α using a photodiode (not shown) or the like. The APC mechanism controls the input energy by a regression circuit, thereby controlling the output of the light source to be constant.

The temperature adjustment mechanism is, for example, a heater or a Peltier element. The wavelength and energy of the outgoing light α emitted from the light source may vary depending on the temperature. For this reason, the wavelength and energy of the outgoing light α emitted from the light source are controlled to be constant by keeping the temperature of the light source constant by the temperature adjusting mechanism.

The angle adjustment mechanism 112 adjusts the incident angle of the outgoing light α with respect to the metal film 30 (the interface between the prism 20 and the metal film 30 (film formation surface 22)). The angle adjusting mechanism 112 emits the emitted light emitted from the light source to irradiate the emitted light α at the first incident angle or the second incident angle toward the predetermined position of the metal film 30 via the prism 20. The optical axis α and the chip holder 142 are relatively rotated. For example, the angle adjustment mechanism 112 rotates the light source unit 111 around the axis perpendicular to the optical axis of the outgoing light α on the metal film 30 (the axis perpendicular to the paper surface of FIG. 2). At this time, the position of the rotation axis is set so that the position of the irradiation spot on the metal film 30 hardly changes even when the incident angle is scanned. In particular, the position of the rotation center is near the intersection of the two optical axes of the emitted light α emitted from the light source at both ends of the scanning range of the incident angle (between the irradiation position on the film formation surface 22 and the incident surface 21). By setting to, the deviation of the irradiation position can be minimized.

The light source control unit 113 controls various devices included in the light source unit 111 to control emission of the emitted light α from the light source unit 111. The light source control unit 113 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.

The light detection unit 120 emits light (for example, fluorescence β, scattered light γ ′ or scattered light emitted from the measurement chip 10 when the light emitting unit 110 irradiates the metal film 30 via the prism 20 on the metal film 30. Plasmon scattered light γ) is detected. In the present embodiment, the light detection unit 120 outputs a signal indicating the detected light amount of the fluorescence β, the light amount of the scattered light γ ′, and the light amount of the plasmon scattered light γ to the control processing unit 150. The light detection unit 120 includes a light receiving optical system unit 121, a position switching mechanism 122, and a sensor control unit 127.

The light receiving optical system unit 121 is disposed on the normal line of the metal film 30 of the measuring chip 10. The light receiving optical system unit 121 includes a first lens 123, an optical filter 124, a second lens 125, and a light receiving sensor 126. The optical axis of the light receiving optical system unit 121 is arranged so as not to coincide with the optical axis of the outgoing light α from the light emitting unit 110. Accordingly, it is possible to prevent the outgoing light α from directly entering the light receiving sensor 126 when detecting the fluorescence β, the scattered light γ ′, or the plasmon scattered light γ. As a result, fluorescence β, scattered light γ ′ or plasmon scattered light γ can be detected with a high S / N ratio.

The position switching mechanism 122 switches the position of the optical filter 124 so that the optical filter 124 is positioned on the optical path in the light receiving optical system unit 121, or a part or all of the optical filter 124 is positioned outside the optical path. Specifically, when the light receiving sensor 126 detects the fluorescence β, the optical filter 124 is disposed on the optical path of the light receiving optical system unit 121, and when the light receiving sensor 126 detects the scattered light γ ′ and the plasmon scattered light γ, A part or all of the optical filter 124 is disposed outside the optical path of the light receiving optical system unit 121.

The first lens 123 is a condensing lens, for example, and condenses light (signal) emitted from the metal film 30. The second lens 125 is, for example, an imaging lens, and forms an image of the light collected by the first lens 123 on the light receiving surface of the light receiving sensor 126. Between both lenses, the light is a substantially parallel light beam.

The optical filter 124 is disposed between the first lens 123 and the second lens 125. The optical filter 124 transmits only the fluorescence component of the light incident on the optical filter 124 and removes the excitation light component (plasmon scattered light γ) during fluorescence detection. Thereby, only the fluorescence component is guided to the light receiving sensor 126, and the fluorescence β can be detected with a high S / N ratio. Examples of types of the optical filter 124 include an excitation light reflection filter, a short wavelength cut filter, and a band pass filter. Examples of the optical filter 124 include a filter including a multilayer film that reflects a predetermined light component, and a color glass filter that absorbs the predetermined light component.

The light receiving sensor 126 detects fluorescence β, scattered light γ ′, and plasmon scattered light γ. By receiving the fluorescence β, the scattered light γ ′, and the plasmon scattered light γ by the same light receiving sensor 126, it is possible to prevent the SPFS device 100 from being enlarged and to reduce the cost. The light receiving sensor 126 is disposed at a position different from the position overlapping the optical axis of the outgoing light α. By detecting the scattered light γ ′ at a position different from the position overlapping with the optical axis of the emitted light α, it is possible to increase the S without detecting the emitted light α that is not scattered in the specimen and emitted onto the measurement chip 10. Scattered light γ ′ can be detected with the / N ratio. The light receiving sensor 126 has a high sensitivity capable of detecting weak fluorescence β from a very small amount of a substance to be measured. The light receiving sensor 126 is, for example, a photomultiplier tube (PMT), an avalanche photodiode (APD), a silicon photodiode (SiPD), or the like.

The sensor control unit 127 controls detection of the output value of the light receiving sensor 126, management of sensitivity of the light receiving sensor 126 based on the output value, change of sensitivity of the light receiving sensor 126 to obtain an appropriate output value, and the like. The sensor control unit 127 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.

The liquid feeding unit 130 supplies the liquid in the liquid chip 50 into the flow path 41 of the measurement chip 10 held by the chip holder 142. In addition, the liquid feeding unit 130 removes the liquid from the flow path 41 of the measurement chip 10. The liquid feeding unit 130 includes a pipette 131 and a pipette control unit 135.

The pipette 131 has a syringe pump 132, a nozzle unit 133 connected to the syringe pump 132, and a pipette tip 134 attached to the tip of the nozzle unit 133. By the reciprocating motion of the plunger in the syringe pump 132, the liquid is sucked and discharged from the pipette tip 134 quantitatively.

The pipette control unit 135 includes a driving device for the syringe pump 132 and a moving device for the nozzle unit 133. The drive device of the syringe pump 132 is a device for reciprocating the plunger of the syringe pump 132, and includes, for example, a stepping motor. The moving device of the nozzle unit 133 moves the nozzle unit 133 freely in the vertical direction, for example. The moving device of the nozzle unit 133 is configured by, for example, a robot arm, a two-axis stage, or a turntable that can move up and down.

The pipette controller 135 drives the syringe pump 132 to suck various liquids from the liquid tip 50 into the pipette tip 134. Then, the pipette controller 135 moves the nozzle unit 133 to insert the pipette tip 134 into the flow channel 41 of the measurement chip 10 and drives the syringe pump 132 to flow the liquid in the pipette tip 134. 41 is injected. In addition, after the introduction of the liquid, the pipette control unit 135 drives the syringe pump 132 to suck the liquid in the channel 41 into the pipette tip 134. By sequentially exchanging the liquid in the channel 41 in this way, the capture body reacts with the substance to be measured in the reaction field (primary reaction), or the measurement substance reacts with the capture body labeled with the fluorescent substance. (Secondary reaction). Further, the liquid feeding unit 130 can dispense or dilute the specimen by sucking or discharging the liquid in the liquid chip 50 as described above.

The transport unit 140 transports and fixes the measurement chip 10. The transport unit 140 includes a transport stage 141 and a chip holder 142.

The transfer stage 141 moves the chip holder 142 in one direction and in the opposite direction. The transport stage 141 has a shape that does not obstruct the optical path of light such as outgoing light α, reflected light of outgoing light α, fluorescence β, scattered light γ ′, and plasmon scattered light γ. The transport stage 141 is driven by, for example, a stepping motor.

The chip holder 142 is fixed to the transfer stage 141 and holds the measurement chip 10 in a detachable manner. The shape of the chip holder 142 can hold the measurement chip 10 and does not obstruct the optical path of light such as the outgoing light α, the reflected light of the outgoing light α, the fluorescence β, the scattered light γ ′, and the plasmon scattered light γ. It is. For example, the chip holder 142 is provided with an opening through which the light passes.

The chip holder 142 is connected to a temperature adjustment mechanism (not shown) such as a heater or a Peltier element. The reaction efficiency in the reaction in the channel 41 such as the primary reaction and the secondary reaction may vary depending on the temperature. For this reason, it is preferable to keep the temperature in the flow path 41 constant by the temperature adjustment mechanism through the chip holder 142, thereby controlling the reaction efficiency constant and increasing the measurement accuracy of the substance to be measured.

The control processing unit 150 controls the angle adjustment mechanism 112, the light source control unit 113, the position switching mechanism 122, the sensor control unit 127, the pipette control unit 135, and the transport stage 141. The control processing unit 150 also functions as a processing unit that processes the detection result of the light detection unit 120 (light receiving sensor 126). In the present embodiment, the control processing unit 150 determines the hematocrit value of the specimen based on the detection result of the scattered light γ ′, and determines the amount of the substance to be measured in the specimen based on the detection result of the fluorescence β. Determine the measured value shown. At the same time, the control processing unit 150 corrects the measurement value based on the hematocrit value, and determines the amount (concentration) of the substance to be measured in plasma or serum. In addition, specific information (for example, data relating to various conversion coefficients, dilution factors, and calibration curves) used in the above processing may be recorded in the control processing unit 150 in advance. In the present embodiment, the control processing unit 150 records in advance a conversion coefficient for correcting (converting) the measured value using the hematocrit value. The control processing unit 150 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.

(Optical path in SPFS device)
As shown in FIG. 2, the outgoing light α enters the prism 20 from the incident surface 21. When the outgoing light α that has entered the prism 20 enters the metal film 30 at the first incident angle, the outgoing light α (first outgoing light α ₁ ) is the liquid and flow in the metal film 30 and the channel 41. Scattered light γ ′ obtained by being scattered by the specimen when passing through the road lid 40 is emitted above the measurement chip 10. Finally, the scattered light γ ′ reaches the light receiving sensor 126. Although not particularly illustrated, a part of the outgoing light α (first outgoing light α ₁ ) is reflected by the metal film 30 to become reflected light, and the reflected light is emitted to the outside of the prism 20 by the outgoing surface 23. Exit.

On the other hand, when the outgoing light α that has entered the prism 20 enters the metal film 30 at a second incident angle that is a total reflection angle at which SPR occurs, localized field light is generated on the metal film 30. This localized field light excites a fluorescent substance that labels the substance to be measured existing on the metal film 30, and emits fluorescence β. The SPFS device 100 detects the fluorescence β emitted from the fluorescent material. Although not particularly illustrated, the reflected light of the emitted light α (second emitted light α ₂ ) from the metal film 30 is emitted to the outside of the prism 20 at the emission surface 23.

(effect)
In the measurement method according to the present embodiment, in order to determine the hematocrit value of the specimen, the first emission light α ₁ is irradiated to the metal film 30 at the first incident angle less than the critical angle. emitted light alpha ₁ detects scattered light gamma 'obtained by being scattered by the sample. Compared with the plasmon scattered light γ, the scattered light γ ′ has a high intensity and a high correlation with the hematocrit value. Therefore, in the measurement method according to the present embodiment, the hematocrit value can be determined with higher accuracy than in the case where plasmon scattered light γ is detected in order to determine the hematocrit value of the specimen. Thereby, in the measuring method according to the present embodiment, the hematocrit value of the specimen can be determined with high accuracy, and the amount (concentration) of the substance to be measured in plasma can be determined with high accuracy. Further, in the measurement method according to the present embodiment, the hematocrit value can be measured with high accuracy without adding a new hematocrit value measurement device, which increases the manufacturing cost and measurement cost of the measurement device. There is nothing.

[Modification]
The measuring method according to the present invention is not limited to the measuring method according to the above embodiment, and the order of each step may be changed as necessary. FIG. 9 is a flowchart illustrating an example of a measurement method according to the modification. For example, as shown in FIG. 9, after the step of detecting the fluorescence signal (step S117), the step of measuring the first optical blank value (step S112) and the step of detecting the scattered light γ ′ (step S115). And may be performed.

The method of performing the above steps S112 and S115 for determining the hematocrit value after detecting fluorescent β is when whole blood having a large dilution ratio, for example, whole blood having a dilution ratio of 10 times or more is used as a specimen. It is a particularly effective means.

In the above embodiment, the sample used in the step of performing the primary reaction and the sample used in the step of detecting the scattered light γ ′ are the same. For this reason, when a specimen having a large dilution rate is used in the primary reaction, the detection accuracy of the scattered light γ ′ is lowered. On the other hand, in the measurement method according to the modified example, in the primary reaction (step S114), even when whole blood having a dilution ratio of 10 times or more is used as a specimen, the step of detecting scattered light γ ′ (step S115). ), A specimen having a smaller dilution ratio (higher concentration) than that of the specimen can be separately prepared and used. As a result, a measurement value indicating the amount of the substance to be measured can be obtained with high accuracy using a specimen with a large dilution ratio (low concentration), and a specimen with a low dilution ratio (high concentration) can be used. Hematocrit value can be obtained with high accuracy. As a result, the amount (concentration) of the substance to be measured in plasma can be determined with higher accuracy.

In the measurement method according to the above-described embodiment, the hematocrit value can be measured using the sample provided in the flow path 41 in the step of performing the primary reaction as compared with the measurement method according to the modification. Therefore, the measurement time of the substance to be measured can be further shortened. In the measurement method according to the above embodiment, after the step of measuring the first optical blank value (step S112), the secondary reaction step (step S116) and the fluorescence signal detection step (step S117) are performed. Therefore, the hematocrit value can be measured with higher accuracy without mixing the fluorescence signal with the first optical blank value.

In the present embodiment, the step of determining the enhancement angle (step S111), the step of measuring the first optical blank value (step S112), the step of measuring the second optical blank value (step S113), and 1 The aspect which performs the process (process S114) which performs a next reaction in this order was demonstrated. However, the measurement method according to the present invention is not limited to this order. For example, the enhancement angle may be determined after performing the primary reaction, or the first optical blank value and the second optical blank value may be measured after performing the primary reaction.

Further, after the step of measuring the first optical blank value (step S112), the step of determining the enhancement angle (step S111) may be performed. However, from the viewpoint of reducing the number of times of switching the position of the optical filter 124 and shortening the measurement time of the substance to be measured, the step of determining the enhancement angle (step S111) and the step of measuring the first optical blank value (step S111) After performing step S112), it is preferable to perform a step of measuring the second optical blank value (step S113).

Further, in the above modification, after the step of performing the primary reaction (step S114), the step of performing the secondary reaction (step S116) was performed (two-step method). However, the timing for labeling the substance to be measured with the fluorescent substance is not particularly limited. For example, before the sample is introduced into the flow channel 41 of the measurement chip 10, a labeling solution may be added to the sample and the substance to be measured may be labeled with a fluorescent substance in advance. Further, the sample and the labeling solution may be injected simultaneously into the flow channel 41 of the measurement chip 10. In the former case, the analyte to be measured is captured by the capturing body by injecting the sample into the flow channel 41 of the measurement chip 10. In the latter case, the substance to be measured is labeled with a fluorescent substance, and the substance to be measured is captured by the capturing body. In any case, both the primary reaction and the secondary reaction can be completed by introducing the sample into the flow channel 41 of the measurement chip 10 (one-step method).

In the above-described embodiment, the SPFS method is used to detect the fluorescent β from the fluorescent substance as a signal. However, the present invention is not limited to this mode. For example, the reflected light of the outgoing light α may be detected as a measurement value using the SPR method.

In the above embodiment, the measurement method including the step of determining the enhancement angle (step S111) has been described. However, the measurement method according to the present invention may not include the step of determining the enhancement angle. In this case, the enhancement angle may be calculated in advance based on factors such as the design of the measurement chip 10 and the refractive index of the liquid provided in the flow path 41.

Furthermore, in the above embodiment, the first outgoing light α ₁ (first light) when detecting scattered light γ ′ from the same light source and the second outgoing light α ₂ when detecting fluorescence β. The mode of irradiating the metal film 30 with (second light) has been described. However, the measurement method according to the present invention is not limited to this mode, and the first outgoing light α ₁ (first light) when detecting the scattered light γ ′ and the second time when detecting the fluorescence β. When the emitted light α ₂ (second light) is irradiated onto the metal film 30, the metal film 30 may be irradiated from different light sources. It is more preferable to use the same light source from the viewpoint of preventing the SPFS device 100 from becoming large and reducing the cost.

This application claims priority based on Japanese Patent Application No. 2016-179396 filed on Sep. 14, 2016. The contents described in the application specification and the drawings are all incorporated herein.

The method for measuring a substance to be measured according to the present invention can detect the substance to be measured with high reliability, and is useful for, for example, examination of diseases.

DESCRIPTION OF SYMBOLS 10 Measurement chip | tip 20 Prism 21 Incident surface 22 Film-forming surface 23 Output surface 30 Metal film 40 Channel cover 41 Channel 50 Liquid chip 100 SPFS apparatus 110 Light emitting part 111 Light source unit 112 Angle adjustment mechanism 113 Light source control part 120 Light detection part REFERENCE SIGNS LIST 121 light receiving optical system unit 122 position switching mechanism 123 first lens 124 optical filter 125 second lens 126 light receiving sensor 127 sensor control unit 130 liquid feeding unit 131 pipette 132 syringe pump 133 nozzle unit 134 pipette tip 135 pipette control unit 140 transport Unit 141 Transport stage 142 Chip holder 150 Control processing unit (processing unit)
α outgoing light α ₁ first outgoing light α ₂ second outgoing light β fluorescence γ plasmon scattered light γ ′ scattered light

Claims

A measurement method for measuring the amount of a substance to be measured in a specimen containing whole blood using surface plasmon resonance,
Preparing a measuring chip having a prism having an incident surface and a film-forming surface, a metal film disposed on the film-forming surface, and a capturing body fixed on the metal film;
In a state where the specimen is present on the metal film, the metal film and the specimen are transmitted when the first light is irradiated from the prism side at a first incident angle less than a critical angle. Detecting the scattered light obtained by scattering the first light in the specimen;
On the metal film, the second substance is emitted from the prism side to the metal film at a second incident angle equal to or greater than a critical angle in a state where the substance to be measured is captured by the capturing body and the sample is not present. A step of detecting a signal indicating the amount of the substance to be measured generated in the measurement chip when irradiated;
Correcting the measured value indicating the amount of the substance to be measured, determined from the detected signal, based on the hematocrit value of the specimen, determined from the amount of the detected scattered light;
Including a measuring method.
The measurement method according to claim 1, wherein the first incident angle is equal to or smaller than an angle smaller than a critical angle by 5 degrees.
The measurement method according to claim 2, wherein the first incident angle is equal to or larger than an angle that is 10 degrees smaller than a critical angle.
When light is irradiated from the prism side while scanning the incident angle, plasmon scattered light generated at the measurement chip is detected, and an enhancement angle that is an incident angle when the light quantity of the plasmon scattered light is maximized is determined. Further comprising a step,
In the step of detecting the signal, the metal film is irradiated with the second light at the enhancement angle.
The measurement method according to any one of claims 1 to 3.
The measurement according to any one of claims 1 to 4, wherein when the metal film is irradiated with P-polarized light at an incident angle of 50 degrees, the light transmittance of the metal film is 3 to 30%. Method.
The measuring method according to claim 5, wherein the metal film has a thickness of 30 to 60 nm.
The measurement method according to any one of claims 1 to 6, wherein the specimen is whole blood having a dilution ratio of 1 to 10.
The measurement method according to claim 7, wherein the specimen is whole blood having a dilution ratio of 1 to 3 times.
The measurement method according to any one of claims 1 to 8, wherein the first light is P-polarized light having a wavelength of 600 to 700 nm.
10. The measurement method according to claim 1, wherein the first light and the second light are applied to the metal film from the same light source.
The measurement method according to claim 10, wherein the first incident angle and the second incident angle are switched by rotating the light source.
The measurement method according to claim 4, wherein the scattered light and the plasmon scattered light are detected by the same light receiving sensor.
The measurement method according to any one of claims 1 to 12, wherein, in the step of detecting the scattered light, the scattered light is detected at a position different from a position overlapping the optical axis of the first light.
14. The measuring method according to claim 1, wherein the signal is fluorescence emitted from a fluorescent substance that labels the substance to be measured.